In many cases, Escherichia coli diarrhea is caused by enterotoxigenic Escherichia coli. The primary causative toxin of Escherichia coli diarrhea is heat-labile toxin (LT) or heat-stable toxin (ST), which a proteinaceous endotoxin produced by enterotoxigenic Escherichia coli. To prevent diseases caused by these bacterial toxins, a method is known in which a vaccine is administered by an injection or a transnasal spray, or administered orally.
Escherichia coli diarrhea is classified into diarrhea during the neonatal period (early-onset diarrhea) and diarrhea after weaning. Preventive measures for these kinds of diarrhea include administration of antibiotics and administration of vaccines. To prevent diarrhea during the neonatal period, inactivated vaccines for immunizing mother pigs have been developed. With the use of such vaccines, suckling pigs are able to receive vaccine antibodies from their mother pigs through milk. In commercially available vaccines which can be obtained domestically, colonization factors such as purified F4, F5 and F6 pili, or inactivated bacterial cells of Escherichia coli expressing these colonization factors are used as immunogens. Some vaccines contain the B subunit of LT, in addition to the above mentioned colonization factors, for the purpose of blocking the binding of LT to cell receptors.
For the prevention of diarrhea after weaning, on the other hand, the amount of immunoglobulin G received from mother pigs via colostrum is considered to be insufficient to protect young pigs, since the half-life of immunoglobulin G in neonatal pigs have been reported to be 13.8 days in average. Although efforts have been made to develop live vaccines which express pili or subunit vaccines comprising pili as components, none of them has been put to practical use. Further, the emergence of Escherichia coli has come to be known in recent years, which produces Stx2e toxin in addition to LT and ST toxins, and more integrated preventive measures are demanded.
Patent Document 1 discloses that the B subunit of Shiga toxin has a vaccine effect against edema disease of swine. Patent Document 2 describes that, when two or three each of Shiga toxin vaccines and cholera toxins are linked for the purpose of improving the production of a recombinant hybrid protein in plants, the amount of the accumulated hybrid protein can be increased. Further, Patent Document 3 discloses the oral administration of plants transformed to express heat-labile toxin and cholera toxin, to carry out immunization.
Non-patent Document 1 discloses findings that the results of experiments in mice suggest that the B subunit of heat-labile toxin may be promising in the prevention of Escherichia coli diarrhea. Non-patent Document 2 describes that heat-labile holotoxin including the B subunit has a vaccine effect against Escherichia coli diarrhea in pigs. Further, Non-patent Document 3 discloses that a hybrid protein in which the B subunits of Shiga toxin are linked can be highly expressed in lettuce.
However, edema disease of swine and Escherichia coli diarrhea generally require different countermeasures in terms of controlling epidemics. Besides, all of the above mentioned prior art documents are silent about the fact that Shiga toxin has an effect of controlling Escherichia coli diarrhea. In addition, although Patent Document 1, 2, and Non-patent Document 3 disclose that it is possible to produce hybrid proteins by introducing Shiga toxin into lettuce or tobacco, it is not described therein that if the hybrid proteins can be produced efficiently in vegetative-propagation plants such as strawberry. Moreover, there are problems that, even if Shiga toxin proteins can be highly expressed in tobacco, they are not suitable in terms of application, because the purification of the proteins and the like are required in order to develop controlling agents and the like, and that it is difficult to maintain acquired recombinant lines over generations, in the case of seed-propagation plants such as tobacco.